A NEW ALKALINE SERINE-PROTEASE FROM ALKALOPHILIC BACILLUS SP - CLONING, SEQUENCING, AND CHARACTERIZATION OF AN INTRACELLULAR PROTEASE

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in whic h there was no signal sequence. The molecular weight was 34,624. The p rotease showed about 50% homology with those of intracellular serine p roteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned i ntracellular protease was expressed in Escherichia coli, and its purif ication and characterization were carried out. The enzyme showed stabi lity under alkaline condition at pH 10 and tolerance to surfactants. T he cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.